Herpes simplex virus-1 (HSV-1) establishes latency in the human peripheral nervous system and provides a textbook example of a stealth pathogen. Viral proteins are not expressed to any significant level and viral transcription is limited to the latency-associated transcripts (LAT) that processed into a stable 2.0-kb intron and handful of viral microRNAs. The absence of latent antigens and lack of discernable phenotypic changes has made it challenging to recognize latently infected neurons without invasive methods that require fixation. Heterologous marker proteins have been uninformative because these are progressively silenced as latency is established. As a result, key questions about the temporal and spatial nature of latency, reactivation and spread within the nervous system remain difficult to answer. This focused 2-year proposal outlines an innovative strategy to construct recombinant HSV-1 viruses that can be readily detected in living neurons during the latent and actively replicating (lytic) stages of the viral lifecycle. Our novel approach uses a ne live cell imaging technology based on short synthetic RNAs (aptamers) that bind a cell-permeable, non-toxic small molecule causing it to emit light in the visible spectrum. To ensure persistent and high-level expression in the neuronal nucleus, this 'green fluorescent RNA' (GFR) will be introduced into the stable 2.0-kb LAT intron. Signal strength and integrity of the engineered viruses will be evaluated using a cultured primary neuron latency/reactivation model developed in our laboratories and subsequently validated in the mouse corneal infection model. Latent infection of neurons with GFR-LAT viruses will produce a distinctive fluorescent signature in latently infected neurons that can be followed in real-time. Additional dual-color viruses will then be constructed by incorporating a red fluorescent protein under the control of a lytic gene promoter. These new viruses will be used to explore the uncertain relationship between levels of LAT expression and propensity of a latent virus to reactivate.